In eukaryotes, the chaperone Hsp90 and its co-chaperones interact with and stabilize a number of crucially important cellular factors, including steroid receptors, kinases and transcription factors. This project will study the interactions of chaperones with these "client" proteins. Despite the recent publication of a number of X-ray studies of Hsp90, the structural states of Hsp90-bound client proteins are completely unknown. Such systems are difficult to crystallize, and even if crystals are formed, structures derived from them may be misleading if significant flexibility is in fact characteristic of these complexes. We propose an NMR spectroscopic approach to this problem, to provide information on these complexes in solution. Some of the protein components are larger than have traditionally been studied by solution NMR, but experiments have been designed that will allow us to draw valid conclusions on the nature of Hsp90-client interactions, using methods recently developed in this laboratory for the analysis of hydrogen/deuterium exchange. This powerful new technique, which employs the aprotic solvent DMSO to trap the labeled species and eliminate scrambling of deuterium label, will be used to probe the structure, stability and interactions of client proteins and co-chaperones with Hsp90 and its domains. Initial validation of the proposed experimental methods will examine the interaction sites of two Hsp90 co-chaperones, p23 and Aha1. Surfaces buried upon complex formation will be detected by comparison of the deuterium-exchange protection of amide protons in samples of 15N-labeled co-chaperone in the presence and absence of chaperone. The optimized methodology will subsequently be applied to the interactions of Hsp90 with client proteins, including the core DNA-binding domain of the tumor suppressor p53, the ligand binding domains of glucocorticoid receptor and estrogen receptor, the PASB domain of hypoxia-inducible factor-2alpha, and the nucleotide binding domain of the cystic fibrosis transmembrane receptor. Binding sites and conformational changes in Hsp90 itself will be further characterized by methyl-specific labeling and high-resolution NMR. During the last few years, interest in the Hsp90 system has increased exponentially, as the central role of Hsp90 in cellular processes is recognized. Nevertheless, there is still very little structural understanding of chaperone-client complexes in solution. This proposal aims to use state-of-the-art NMR methods to address this problem. PUBLIC HEALTH RELEVANCE: The chaperone Hsp90 is involved in critically important cellular functions, including stabilizing the structures of client proteins such as transcription factors and kinases, and aiding in the stabilization and transport of proteins across membranes. Hsp90 inhibitors are the subject of intense study in the pharmaceutical industry, as general therapies for cancers, and Hsp90 and its co-chaperones also appear to be involved with proteins that give rise to diseases such as cystic fibrosis. Our studies will elucidate the structural and dynamic nature of these complexes in solution, as a guide for future drug design efforts. [unreadable] [unreadable] [unreadable]